Although 2% glutaraldehyde is often the first-line agent for endoscopic disinfection, its adverse reactions are common among staff and it is less effective against certain mycobacteria and spore-bearing bacteria. Chlorine dioxide is a possible alternative and an automated washer-disinfector fitted with this agent is currently available. This study was conducted to evaluate the effectiveness of chlorine dioxide in endoscopic disinfection after upper gastrointestinal examination. In vitro microbicidal properties of chlorine dioxide solutions were examined at high (600 ppm) and low (30 ppm) concentrations against various microbes including Pseudomonas aeruginosa, Helicobacter pylori, Mycobacterium avium-intracellulare and Bacillus subtilis in the presence or absence of bovine serum albumin (BSA). Immediately following endoscopic procedures and after application to the automated reprocessor incorporating chlorine dioxide at 30 ppm for 5 min, endoscopic contamination with infectious agents, blood, H. pylori ureA gene DNA and HCV-RNA was assessed by cultivation, sensitive test tape, polymerase chain reaction (PCR) and reverse transcriptase-PCR analysis, respectively. Chlorine dioxide at 30 ppm has equivalent microbicidal activity against most microbes and faster antimicrobial effects on M. avium-intracellulare and B. subtilis compared with 2% glutaraldehyde, but contamination with BSA affected the microbicidal properties of chlorine dioxide. Endoscopic contamination with microbes, blood and bacterial DNA was eliminated after application of the automated reprocessor/chlorine dioxide system. Thus, chlorine dioxide is a potential alternative to glutaraldehyde. The use of automated reprocessors with compatibility to chlorine dioxide, coupled with thorough pre-cleaning, can offer effective, faster and less problematic endoscopic disinfection.